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首页> 外文期刊>Journal of bacteriology >Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp.
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Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp.

机译：新型IS3基团插入元件的分离和乳酸杆菌属的整合载体的构建。

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An insertion sequence (IS) element from Lactobacillus johnsonii was isolated, characterized, and exploited to construct an IS-based integration vector. L. johnsonii NCK61, a high-frequency conjugal donor of bacteriocin production (Laf+) and immunity (Lafr), was transformed to erythromycin resistance (Emr) with the shuttle vector pSA3. The NCK61 conjugative functions were used to mobilize pSA3 into a Laf- Lafs EMs recipient. DNA from the Emr transconjugants transformed into Escherichia coli MC1061 yielded a resolution plasmid with the same size as that of pSA3 with a 1.5-kb insertion. The gram-positive replication region of the resolution plasmid was removed to generate a pSA3-based suicide vector (pTRK327) bearing the 1.5-kb insert of Lactobacillus origin. Plasmid pTRK327 inserted randomly into the chromosomes of both Lactobacillus gasseri ATCC 33323 and VPI 11759. No homology was detected between plasmid and total host DNAs, suggesting a Rec-independent insertion. The DNA sequence of the 1.5-kb region revealed the characteristics of an IS element (designated IS1223): a length of 1,492 bp; flanking, 25-bp, imperfect inverted repeats; and two overlapping open reading frames (ORFs). Sequence comparisons revealed 71.1% similarity, including 35.7% identity, between the deduced ORFB protein of the E. coli IS element IS150 and the putative ORFB protein encoded by the Lactobacillus IS element. A putative frameshift site was detected between the overlapping ORFs of the Lactobacillus IS element. It is proposed that, similar to IS150, IS1223 produces an active transposase via translational frameshifting between two tandem, overlapping ORFs.
机译：分离，表征了来自约翰逊乳杆菌的插入序列（IS）元件，并对其进行了构建以构建基于IS的整合载体。约翰逊L. johnsonii NCK61是细菌产生（Laf +）和免疫（Lafr）的高频共生供体，用穿梭载体pSA3转化为对红霉素的抗性（Emr）。使用NCK61的结合功能将pSA3动员到Laf-Lafs EM受体中。将来自Emr转导结合体的DNA转化到大肠杆菌MC1061中，得到的分离质粒大小与pSA3相同，插入长度为1.5 kb。去除分离质粒的革兰氏阳性复制区，以产生带有1.5-kb插入乳杆菌来源的基于pSA3的自杀载体（pTRK327）。质粒pTRK327随机插入加氏乳杆菌ATCC 33323和VPI 11759的染色体中。在质粒和总宿主DNA之间未检测到同源性，这表明与Rec无关。 1.5kb区域的DNA序列显示出IS元件（命名为IS1223）的特征：长度为1,492bp;以及侧翼的25 bp不完美的反向重复序列;和两个重叠的开放阅读框（ORF）。序列比较显示，推导的大肠杆菌IS元件IS150的ORFB蛋白与由乳杆菌IS元件编码的假定的ORFB蛋白之间有71.1％的相似性，包括35.7％的同一性。在乳杆菌IS元件的重叠ORF之间检测到一个假定的移码位点。提议与IS150相似，IS1223通过两个串联，重叠的ORF之间的翻译移码产生活性转座酶。

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